Absolute Cell Numbers Research Articles

The Comprehensive Morphological Criteria for the Diagnosis of Subclinical Bacterial Maternal-Fetal Infection in Offspring

The paper presents study materials devoted to determining the morphofunctional state of progeny’s organs developed under subclinical bacterial maternal-fetal infection (MFI) in order to define the comprehensive diagnostic criteria of this pathology. The rat models of subclinical MFI caused separately by E. coli, S. aureus, and K. pneumoniae were used, the set of diagnostic tools (histopathological staining of paraffin sections of aorta (AO), pulmonary artery (PA), thyroid (TG), and adrenal glands (AG), liver (LV) of fetuses with H&E, Mallory’s trichrome and Van Gieson's methods, optical microscopy, morphometry, and immunofluorescence with the use of ImageJ software) was applied. Statistical analysis was performed in Microsoft Excel 365 and R. Depending on the organ, the null hypothesis was rejected when p<0.05 or p<0.001. The assemblage of statistically significant diagnostic parameters of the subclinical MFI damaging effect in offspring’s organism was determined: for AO and PA – endotheliocyte’s height, the optical density of fluorescence (ODF) of CD34-positive cells, type III and IV collagens; for TG – thyrocyte’s height, nuclear-cytoplasmic ratio (NCR), follicle’s square, T4 OD; for AG – adrenocorticocytes’ density in zona glomerulosa, adrenocorticocyte’s NCR, cortisol ODF in zona fasciculata; for LV - hepatocytes’ density, NCR, the absolute number of IL-6 producing cells. Conclusion: in this study, we have proved the presence of pathomorphological substrate and defined dynamics of morphofunctional changes forming in the fetal organism under the bacterial MFI that enables the use of obtained values as diagnostic criteria of this pathology.

Decrease of peripheral blood mucosal\u2010associated invariant T cells and impaired serum Granzyme-B production in patients with gastric cancer

Mucosal-associated invariant T (MAIT) cells are an invariant T cell subset, which have been reported to play an antimicrobial role in infectious diseases. However, little is known about it in malignant diseases and tumors, especially in gastric cancer (GC). So in this study, we aim to examine the frequency, phenotype, partial functional capacity and clinical relevance of this cells from GC patients’ peripheral blood by flow cytometry. It was shown that the frequency of peripheral blood MAIT cells was negatively correlated with their increasing age in healthy adults. Importantly, comparing to the healthy controls (HC), the frequency and the absolute number of MAIT cells from GC patients’ peripheral blood with or without chemotherapy were both significantly lower than those. For the phenotype, the proportion of CD4−MAIT cell subset in GC patients without chemotherapy was lower than in HC, but higher than in GC patients with chemotherapy. Whereas, the proportion of CD4−CD8+MAIT cell subset in GC patients without chemotherapy was significantly lower than that in HC. Finally, the level of Granzyme-B (GrB), a molecule associated with MAIT cells was markedly lower in GC patients. But the correlation between the serum levels of GC-associated tumor antigens and the percentages of MAIT cells in GC patients was not observed. In conclusion, our study shows the decreased frequency, changed phenotypes and partial potentially impaired function of MAIT cells in GC patients, suggesting a possible MAIT cell-based immunological surveillance of GC.

Four Biomarkers Linked to Activation of Cluster of Differentiation 8-Positive Lymphocytes Predict Clinical Outcomes in Pediatric Acute Liver Failure.

Immune dysregulation contributes to the pathogenesis of pediatric acute liver failure (PALF). Our aim was to identify immune activation markers (IAMs) in PALF that are associated with a distinct clinical phenotype and outcome. Among 47 PALF study participants, 12 IAMs collected ≤6days after enrollment were measured by flow cytometry and IMMULITE assay on blood natural killer and cluster of differentiation 8-positive (CD8+ ) lymphocytes and subjected to unsupervised hierarchical analyses. A derivation cohort using 4 of 12 IAMs which were available in all participants (percent perforin-positive and percent granzyme-positive CD8 cells, absolute number of CD8 cells, soluble interleukin-2 receptor level) were sufficient to define high (n=10), medium (n=15), and low IAM (n=22) cohorts. High IAM was more frequent among those with indeterminate etiology than those with defined diagnoses (80% versus 20%, P<0.001). High IAM was associated with higher peak serum total bilirubin levels than low IAM (median peak 21.7 versus 4.8mg/dL, P<0.001) and peak coma grades. The 21-day outcomes differed between groups, with liver transplantation more frequent in high IAM participants (62.5%) than those with medium (28.2%) or low IAM (4.8%) (P=0.002); no deaths were reported. In an independent validation cohort (n=71) enrolled in a prior study, segregation of IAM groups by etiology, initial biochemistries, and short-term outcomes was similar, although not statistically significant. High serum aminotransferases, total bilirubin levels, and leukopenia at study entry predicted a high immune activation profile. Four circulating T-lymphocyte activation markers identify a subgroup of PALF participants with evidence of immune activation associated with a distinct clinical phenotype and liver transplantation; these biomarkers may identify PALF participants eligible for future clinical trials of early targeted immunosuppression.

Persistence of Epstein-Barr virus antigens in gastric cancer: characteristic of inflammatory cellular reactions in tumor

Is to establish the relationship between the persistence of viral antigens of the Epstein-Barr virus (EBV) and the cellular composition of the immune microenvironment of tumor tissue and the mucous membrane of peritumoral area in gastric cancer. We used samples of surgical material from 55 patients with a verified diagnosis of gastric cancer. The expression of CD4, CD8, CD68, CD1a and LMP-1 was assessed. The results were assessed by the morphometric method. We selected three fields of view (magnification x200) in tumor tissue and in peritumoral areas separately and counted an absolute number of cells with positive staining with further calculation of the average number of cells and the median. LMP-1-negative tumors with LMP-1 expression in epithelium of peritumoral area were characterized by the highest density of CD4+ lymphocyte infiltration in the central part of the tumor; the highest density of CD8+ lymphocyte infiltration in the mucous membrane of peritumoral area (p=0.0190); the highest density of infiltration by macrophages in the mucous membrane of peritumoral area (p=0.2492); the highest density of infiltration by CD1a+ cells in the mucous membrane of peritumoral area (p=0.1503). The highest density of infiltration with CD1a+ cells was characteristic for LMP-1-positive and LMP-1-negative tumors (p=0.0813). The persistence of the LMP-1 viral antigen in the glandular epithelium of the peritumoral area in our sample does not have a statistically significant effect on the prognosis of the disease (RR=1.7718; p=0.0885) but there is a tendency towards a negative predictive value. High density of infiltration of glandular epithelium of peritumoral area with the expression of LMP-1 by CD4+ and CD8+ lymphocytes is most likely associated with the activation of the cellular immune response and may be one of the signs of the persistence of viral antigens. It was shown for the first time that the phenomenon of persistence of the LMP-1 viral antigen is characterized by a trend towards negative predictive value for patients with gastric cancer.

Acetylsalicylic acid (Aspirin®) and liver regeneration: experimental study in rats.

to evaluate the influence of acetylsalicylic acid (ASA) on cell proliferation after partial hepatectomy in rats. 40 male Wistar rats were separated into four groups of ten rats each. Groups 1 and 2 (controls): undergoing 30% partial hepatectomy and, after one day (group 1) and seven days (group 2), to euthanasia; daily administration of 0.9% saline solution (1mL per 200g of body weight). Groups 3 and 4 (experimental): undergoing 30% partial hepatectomy and, after one day (group 3) and seven days (group 4), to euthanasia; daily administration of ASA (40mg/mL, 1mL per 200g of body weight). The absolute number of cells stained with PCNA was counted in photomicrographs, in five fields, and it was calculated the mean of positive cells per animal and per group. the final mean of PCNA+ cells per group was: in group 1, 17.57 ± 6.77; in group 2, 19.31 ± 5.30; in group 3, 27.46 ± 11.55; and, in group 4, 12.40 ± 5.23. There was no significant difference at the two evaluation times in the control group (p=0.491), but there was in the experimental group (p=0.020), with a lower number of PCNA+ cells on the seventh day. The comparison between the two groups, on the first day, showed more PCNA+ cells in the livers of the animals that received ASA (p=0.047), and on the seventh day the number was lower in the experimental group (p=0.007). ASA induced greater hepatocyte proliferation.

Sepsis promotes gliogenesis and depletes the pool of radial glia like stem cells in the hippocampus

Sepsis associated encephalopathy (SAE) is a major complication of patients surviving sepsis with a prevalence up to 70%. Although the initial pathophysiological events of SAE are considered to arise during the acute phase of sepsis, there is increasing evidence that SAE leads to persistent brain dysfunction with severe cognitive decline in later life. Previous studies suggest that the hippocampal formation is particularly involved leading to atrophy in later stages. Thereby, the underlying cellular mechanisms are only poorly understood. Here, we hypothesized that endogenous neural stems cells and adult neurogenesis in the hippocampus are impaired following sepsis and that these changes may contribute to persistent cognitive dysfunction when the animals have physically fully recovered. We used the murine sepsis model of peritoneal contamination and infection (PCI) and combined different labeling methods of precursor cells with confocal microscopy studies to assess the neurogenic niche in the dentate gyrus at day 42 postsepsis. We found that following sepsis i) gliogenesis is increased, ii) the absolute number of radial glia-like cells (type 1 cells), which are considered the putative stem cells, is significantly reduced, iii) the generation of new neurons is not significantly altered, while iv) the synaptic spine maturation of new neurons is impaired with a shift to expression of more immature and less mature spines.In conclusion, sepsis mainly leads to depletion of the neural stem cell pool and enhanced gliogenesis in the dentate gyrus which points towards an accelerated aging of the hippocampus due to septic insult.

Early Adaptive Natural Killer Cell Expansion Is Associated with Decreased Relapse After Autologous Transplantation for Multiple Myeloma

Adaptive natural killer (NK) cells are long-lived and exhibit properties of immunologic memory against cytomegalovirus (CMV). We previously reported that expansion of adaptive NK cells after CMV reactivation in recipients of allogeneic hematopoietic cell transplantation (HCT) was associated with a lower rate of relapse of acute myelogenous leukemia. In the present study, we examined the impact of adaptive NK cell expansion in a cohort of 110 individuals who underwent autologous HCT (AHCT) for a lymphoid malignancy (lymphoma or multiple myeloma [MM]). In this cohort, higher absolute numbers of adaptive NK cells (>1.58/μL) at day 28 post-AHCT were associated with significantly decreased risk of relapse in patients with MM. No significant association was seen in patients with lymphoma. Further stratification of MM patients by CMV serostatus found a strong protective effect of adaptive NK cells only in CMV-seropositive individuals. These findings suggest that strategies to increase adaptive NK cells after AHCT may be a therapeutic option in patients with MM.

The Impact of Age and Gender on Hematopoietic Stem Cells and Immune Contexture of the Bone Marrow Microenvironment

Hematopoietic stem cells (HSCs) are a rare population of cells that reside mainly in the bone marrow and are capable of generating and fulfilling the entire hematopoietic system upon differentiation. Thirty-six healthy donors, attending the HSCT center to donate their bone marrow, were categorized according to their age into child (0–12 years), adolescence (13–18 years), and adult (19–59 years) groups, and gender into male and female groups. Then, the absolute number of HSCs and mature immune cells in their harvested bone marrow was investigated. Here, we report that the absolute cell number can vary considerably based on the age of the healthy donor, and the number of both HSCs and immune cells declines with advancing age. The gender of the donor (male or female) did not have any impact on the number of the HSCs and immune cells in the bone marrow. In conclusion, since the number of HSCs plays a pivotal role in the clinical outcome of allogeneic HSC transplantations, identifying a younger donor regardless the gender is critical.

A Mineralocorticoid Receptor Deficiency in Myeloid Cells Reduces Liver Steatosis by Impairing Activation of CD8+ T Cells in a Nonalcoholic Steatohepatitis Mouse Model.

Background and AimsThe mineralocorticoid receptor (MR) and renin-angiotensin-aldosterone system (RAAS) are implicated in non-alcoholic liver fatty disease (NALFD). However, inflammatory mechanisms linking MR and RAAS with disease pathology remain unclear. Here we aimed to evaluate the contribution of myeloid MR to the inflammatory response in an animal model of non-alcoholic steatohepatitis (NASH), induced with a methionine-choline deficient diet (MCD).MethodsMice with a conditional deficiency of MR in myeloid cells (MyMRKO) and their counterpart floxed control mice (FC) were fed for 18 days with MCD or chow diet, respectively. Serum levels of aminotransferases and aldosterone levels were measured and hepatic steatosis, inflammation and fibrosis scored histologically. Hepatic triglyceride content (HTC) and hepatic mRNA levels of pro-inflammatory pro-fibrotic-associated genes were also assessed. Deep flow cytometric analysis was used to dissect the immune response during NASH development.ResultsMyMRKO mice fed with an MCD diet exhibited reduced hepatic inflammation and lower HTC than controls. Absolute number and percentage of liver inflammatory infiltrate cells (except for CD8+ T lymphocytes) were similar in both MyMRKO and control mice fed with an MCD diet but expression of the costimulatory molecule CD86 by dendritic cells and the CD25 activation marker in CD8+ T cells were significantly reduced in MyMRKO.ConclusionsProinflammatory cells are functionally suppressed in the absence of MR. We hypothesized that loss of MR in myeloid cells reduces lipid accumulation in the liver, in part through modulating the adaptive immune response, which is pivotal for the development of steatosis.

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center

BackgroundThis study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the AccuriTM C6 cytometer in providing direct counts of absolute cell numbers. MethodWe evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer AccuriTM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the AccuriTM C6. Main ResultsIn our analysis, the results showed a strong correlation between the number of CD34+ cells/μL (r2=0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/μL cells (r2=0.64). ConclusionOur results showed that the AccuriTM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

Age-Related Dynamics of Circulating Innate Lymphoid Cells in an African Population

Innate lymphoid cell (ILC) lineages mirror those of CD4+ T helper cell subsets, producing type 1, 2 and 3 cytokines respectively. Studies in adult human populations have shown contributions of non-cytotoxic ILC to immune regulation or pathogenesis in a wide range of diseases and have prompted investigations of potential functional redundancy between ILC and T helper cell compartments in neonates and children. To investigate the potential for ILC to contribute to immune responses across the human lifespan, we examined the numbers and frequencies of peripheral blood ILC subsets in a cohort of Gambians aged between 5 and 73 years of age. ILC2 were the most abundant peripheral blood ILC subset in this Gambian cohort, while ILC1 were the rarest at all ages. Moreover, the frequency of ILC1s (as a proportion of all lymphocytes) was remarkably stable over the life course whereas ILC3 cell frequencies and absolute numbers declined steadily across the life course and ILC2 frequencies and absolute numbers declined from childhood until the age of approx. 30 years of age. Age-related reductions in ILC2 cell numbers appeared to be partially offset by increasing numbers of total and GATA3+ central memory (CD45RA-CCR7+) CD4+ T cells, although there was also a gradual decline in numbers of total and GATA3+ effector memory (CD45RA-CCR7-) CD4+ T cells. Despite reduced overall abundance of ILC2 cells, we observed a coincident increase in the proportion of CD117+ ILC2, indicating potential for age-related adaptation of these cells in childhood and early adulthood. While both CD117+ and CD117- ILC2 cells produced IL-13, these responses occurred predominantly within CD117- cells. Furthermore, comparison of ILC frequencies between aged-matched Gambian and UK young adults (25–29 years) revealed an overall higher proportion of ILC1 and ILC2, but not ILC3 in Gambians. Thus, these data indicate ongoing age-related changes in ILC2 cells throughout life, which retain the capacity to differentiate into potent type 2 cytokine producing cells, consistent with an ongoing role in immune modulation.

Clinical Characteristics and Outcomes of Patients with Large Granular Lymphocytic Leukemia Treated with Methotrexate, Cyclophosphamide or Cyclosporine. a Large Single Institutional Retrospective Analysis

Introduction Large granular lymphocytic leukemia (LGLL) is a rare mature clonal lymphoproliferative disorder derived from T-cell or natural killer (NK)-cell lineages. Even though LGLL has usually an indolent course, a majority of patients (pts) require therapy due to cytopenias, recurrent infections, autoimmune disorders or symptomatic splenomegaly. There is no standard frontline therapy established for pts with LGLL based on randomized prospective studies but a single-agent immunosuppressive therapy has been used for almost four decades. In the present study, we analyzed clinical and pathological characteristics, responses to therapy and outcomes of patients diagnosed at Moffitt Cancer Center. Methods A total of 303 consecutive LGLL pts diagnosed between 2001 and 2017 were included in the study. Pts who did not meet the WHO diagnostic criteria of peripheral blood (PB) LGL count ≥2.0 k/ μL but presented with smaller, persistent LGL clonal populations along with characteristic immunophenotype and clinical features such as cytopenias, recurrent infections, autoimmune disorders, splenomegaly, and bone marrow involvement with identical clonal LGL population were considered to have LGLL. Responses to treatment were categorized as complete response (CR), partial response (PR) and no response. CR was defined as Hb&amp;gt;12g/dL, ANC≥1.5k/μL, PLT&amp;gt;150k/μL ALC&amp;lt;4 k/μL and LGL&amp;lt;0.5K/μL. PR was defined as Hb&amp;gt;8g/dL, ANC&amp;gt;0.5K/μL, PLT&amp;gt;50k/μL, and no transfusion requirement. Cox proportional hazard mode was used for multivariate survival analysis with all the covariates listed in table 3. Results The median age at diagnosis was 65 years (range: 21 - 90 years). 92.1% of pts were diagnosed with T-LGLL and 7.9% with NK-LGLL. At presentation, 50.4% of pts had anemia; 48.4% had neutropenia and 34.6% had thrombocytopenia. 21.7% of pts were transfusion dependent; 20.3% had severe neutropenia (ANC &amp;lt; 0.5K/μL) and 33% had splenomegaly. Preexisting diagnosis of autoimmune disorders was observed in 26.4% of pts (Table 1). The Foundation One Heme NGS assay (Foundation Medicine Inc, Cambridge, Massachusetts, USA) was employed in 25 pts (www.foundationmedicine.com/genomic-testing/foundation-one-heme). Nine of 25 pts were positive for STAT 3 and none for STAT5 mutations. 54.8% of pts required treatment. Table 2 summarizes the first three lines of therapy. Methotrexate (MTX), cyclophosphamide, and cyclosporine A (CSA) were the most commonly used front-line therapies. Overall response (CR + PR) for all 3 agents ranged between 55%-67% with CR rate of 11%-25%. 25% of pts receiving Cytoxan achieved CR and 11.7% on MTX achieved CR however statistical testing was not significant due to the limitation of sample size. Rates of overall response (OS) were not different among the treatment regiments. The rate of 10-year overall survival (OS) was 56.8%. Median OS was 14 years [95% CI (9.7 years, .) (Figure 1). In the multivariate Cox model, increasing age, neutropenia, thrombopenia and coexisting malignancies were independent factors associated with poor survival. Absolute number of LGLL cells in blood was not associated with OS. T-LGLL and NK-LGLL had comparable OS (Table 3). Conclusions This study is one of the largest retrospective single-institutional studies of LGLL. Consistent with prior studies, LGLL demonstrated an indolent course with a long median OS. ORR and CR rates did not significantly differ among cyclophosphamide, MTX and CSA. However, a significant percentage of patients did not respond to the frontline immunosuppressive therapy. Clinical trials with novel therapeutic agents are necessary to further improve outcomes of patients with LGLL. Disclosures Komrokji: Jazz: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Agios: Speakers Bureau; Abbvie: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Novartis: Honoraria; Geron: Honoraria. Sokol:EUSA Pharma: Consultancy, Honoraria, Speakers Bureau; Kyowa/Kirin Inc.: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Systems-Level Analysis of the Immune Repertoire in Neutropenia Reveal Arrested NK Cell Differentiation and Exhaustion

Neutrophils are innate cells that have been suggested to play a critical role in terminal differentiation of NK cells. Whether this is a direct effect or a consequence of global immune changes with effects on NK cell homeostasis remains unknown. Here, we used high-resolution flow and mass cytometry to examine NK cell repertoires in 64 neutropenic patients and 27 healthy age- and gender-matched controls. A subgroup of neutropenic patients had lower frequencies and absolute numbers of NK cells, yet increased frequencies of CD56bright among NK cells (Figure 1A-C). Moreover, their CD56dim compartment was characterized by a block in differentiation, with a relative lack of NKG2A-CD57+KIR+ NK cells. In line with the differentiation arrest, no expansion of adaptive NK cells could be detected in CMV-seropositive patients from this subgroup. Furthermore, CD56dim NK cells showed increased frequencies of Ki-67+, Tim-3+ and TIGIT+ cells suggestive of activation and exhaustion (Figure 1D). The systemic imprint in the NK cell repertoire was associated with a blunted tumor target cell response with inefficient killing and a lower proportion of degranulating CD56dim cells (Figure 1E). RNA sequencing of the NK cell compartment further revealed that the differentiation arrest was linked to increased expression of transcription factors and genes involved in proliferation and cytokine signaling (Figure 1F). Serum protein profiling of 264 proteins showed upregulation of pathways related to apoptosis and cell turnover, as well as immune regulation and inflammation including higher levels of IL-10, IL-18 and IL-27 in these patients (Figure 1G-H). Notably, the majority of patients with perturbed NK cell compartment exhibited high-grade neutropenia, overall suggesting that the profoundly altered NK cell homeostasis was tightly connected to the severity of their underlying etiology (Figure 1I). Disclosures Meinke: XNK Therapeutics AB: Consultancy. Palmblad:Roche Sweden Inc: Speakers Bureau; Chieti Canada Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents &amp; Royalties.

GTB-3550 TriKE™ for the Treatment of High-Risk Myelodysplastic Syndromes (MDS) and Refractory/Relapsed Acute Myeloid Leukemia (AML) Safely Drives Natural Killer (NK) Cell Proliferation At Initial Dose Cohorts

Introduction: Relapsed/refractory AML and MDS present a clinical challenge. Despite FDA approval of multiple new targeted agents, many patients lack actionable mutations and have exhausted conventional chemotherapeutic options. We have shown that NK cell infusions after lymphodepleting chemotherapy can induce remissions in relapsed/refractory AML. However, this cell-based therapy lacks antigen specificity. To address this, we developed GTB-3550 TriKE, a novel CD16/IL-15/CD33 Tri-Specific Killer Engager (TriKE). GTB-3550 TriKE is comprised of two single chain variable fragments (scFvs); one that binds CD16 on NK cells and one that binds CD33 on myeloid malignancies, plus an IL-15 linker bridging the CD16 and CD33 scFvs for sustained cell activation. Pre-clinical data shows specific NK cell activation and targeted cytolytic activity in xenogeneic AML mouse models. Methods: Patients with CD33+ malignancies (primary induction failure or relapsed AML with failure of one reinduction attempt or high risk MDS progressed on two lines of therapy) age 18 and older were eligible (NCT03214666) if they had adequate renal, hepatic, cardiac and lung function and absolute lymphocyte count (ALC) ≥ 200 cells/µL or absolute circulating CD56+/CD3- NK cell count &amp;gt;25 cells/µL within the 14 days prior to start of therapy. The primary endpoint is to identify the maximum tolerated dose (MTD) of GTB-3550 TriKE. Correlative objectives include the number, phenotype, activation status and function of NK cells and T cells. During phase 1, each patient received the GTB-3550 TriKE at 5-200 mcg/kg/day (in 6 cohorts) for 3 weeks (infusion block #1, #2, and #3) of 96 hours continuous infusion separated by a 72-hour rest. Two patients enrolled per dose cohort. Disease response assessed by bone marrow biopsy between Days 21-42. Results: Four patients have enrolled, two at 5 mcg/kg/day and two at 10 mcg/kg/day, and three have completed therapy. Two patients with stable disease and one had substantial disease progression in the setting of a FLT-3 ITD mutation (Figure 1A). The first patient at 5 mcg/kg/day had stable disease after course 1 and qualified for retreatment with a second course of 3 weeks of GTB-3550 TriKE due to improved platelet transfusion requirements. Of the four complete courses of treatment delivered, all were delivered on schedule without treatment interruptions. The previous MTD of continuous infusion rhIL-15 was 2 mcg/kg/day (Conlon et al, Clin. Ca Res.) with associated fevers, tachycardia and constitutional symptoms. Validating our pre-clinical data showing decreased IL-15 potency when sandwiched between the CD16 and CD33 scFvs, patients treated with GTB-3550 TriKE treatment displayed no signs of clinical immune activation or SAE's in the 5 and 10 mcg/kg/day cohorts. Correlative studies have shown reproducible NK cell activity in all patients. NK cell activation (CD69+) increases early during treatment (Figure 1B). The greatest NK cell proliferation (Ki-67+) starts at day 3, is maximal at Day 8, and maintained above baseline at Day 15 and Day 22 (Figure 1C). This correlated with an increase proportion and absolute number of NK cells during treatment (Figure 1D). Targeted delivery of IL-15 to NK cells through the anti-CD16 engager showed preferential proliferation of NK cells and significantly less effect on CD8+ T cells. Additionally, the frequency of NK cells and absolute lymphocyte count decreased early during the continuous infusion of the GTB-3550 TriKE, which rebounded after the 72-hour rest period to higher levels than the pre-treatment baseline. Conclusion: This novel GTB-3550 TriKE administered by continuous infusion led to NK cell proliferation in all patients at initial dose levels and no clinically significant targeted toxicity. Though no objective responses were seen at the initially low dose cohorts tested, these early data suggest proof of principle that the drug has immune activity in humans. Subsequent patients given higher dose levels will determine if NK cell proliferation translates into clinical efficacy. Disclosures Weisdorf: Incyte: Research Funding; FATE Therapeutics: Consultancy. Vallera:GT Biopharma: Consultancy, Patents &amp; Royalties, Research Funding. Schroeder:GT Biopharma: Consultancy, Current equity holder in publicly-traded company. Felices:GT Biopharma: Consultancy. Miller:Fate Therapeutics, Inc: Consultancy, Patents &amp; Royalties, Research Funding; GT Biopharma: Consultancy, Patents &amp; Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: GTB-3550 TriKE is a cytokine immune engager binding to CD16 on NK cells, CD33 on myeloid blasts and IL-15 between the two engager components.

Muse cells, endogenous reparative pluripotent stem cells, are mobilized into the peripheral blood after percutaneous coronary intervention in patients with coronary artery disease

Abstract Background Multilineage-differentiating stress enduring (Muse) cells, endogenous reparative pluripotent stem cells, are defined as stage-specific embryonic antigen 3+ (SSEA3+) and CD105+ double-positive cells. We previously reported that intravenously injected Muse cells home to the damaged heart and differentiate into cardiomyocytes and vessels, and reduce the infarct size and improve cardiac function in a rabbit model of acute myocardial infarction (AMI) (Circ Res 2018), and that endogenous Muse cells are mobilized into the peripheral blood in the acute phase of patients with AMI, which improve left ventricular (LV) function and attenuate LV remodeling in the chronic phase at 6 months (Circ J 2018). However, whether Muse cells are mobilized into the peripheral blood after percutaneous coronary intervention (PCI) in patients with coronary artery disease. Methods Muse cells in the peripheral blood was measured by fluorescence-activated cell sorting (FACS) as SSEA3+ and CD105+ double-positive cells in patients with coronary artery disease with 75% coronary stenosis who underwent PCI (n=18) with a mean age of 73.0±7.2 (14 male and 4 female). Blood samples were collected from the antecubital vein in patients with coronary artery disease before, and 1 and 24 h after PCI. Since the majority of Muse cells were detected in the monocyte area and few Muse cells if any were detected in the lymphocyte area, we counted the Muse cells in the monocyte area by FACS. The number of Muse cells was expressed as cells per 100 μL of blood, as follows: absolute number of Muse cells (/100 μL) = white blood cells (/100 μL) × monocytes (%) × SSEA3+/CD105+ double-positive cells (%). Results Typical case of SSEA3+/CD105+ double-positive Muse cells measured by FACS shows that majority of Muse cells exist in the monocyte area (Fig, 1-A). The number of Muse cells in the peripheral blood was significantly greater (p&amp;lt;0.05) at 1 h (58.6±23.8 /100 μL) or 24 h after PCI (69.7±43.1/100 μL) as compared with that before PCI (46.3±19.0/100 μL) (Fig. 1-B). Conclusion Muse cells, endogenous pluripotent stem cells, are mobilized into the peripheral circulating blood 1 h and 24 h after PCI in patients with coronary artery disease. Mobilized Muse cells after PCI might be contributing to repair the damaged coronary artery. Figure 1 Funding Acknowledgement Type of funding source: None

Epicardial adipose tissue CD4+ T cells as novel drivers in the development of atrial fibrillation

Abstract Background Epicardial adipose tissue (EAT) is now a well-established independent risk factor for all forms of atrial fibrillation (AF). However, the cellular profile and immune mediators within EAT remain poorly defined. Purpose This study sought to define the immunological signature of EAT critical to its role in the development of both persistent AF and de novo post-operative AF (POAF). Methods Adult patients undergoing elective cardiac surgery with no prior AF history and those with pre-existing AF were recruited to undergo EAT sampling alongside subcutaneous adipose tissue (SAT) and pre-operative blood (adipose tissue and systemic controls respectively) sampling. The blood and tissue samples were immediately taken to the laboratory for immune cell isolation, flow cytometry and quantitative polymerase chain reaction (qPCR) analysis. Results A total of 71 patients were recruited including 9 patients with a prior history of AF. Of the 62 a priori sinus rhythm (SR) patients, 42 remained in SR post-operatively and 20 developed de novo POAF. No differences in absolute numbers of immune cells in the blood, EAT or SAT were detected on flow cytometry analysis between the three groups of patients. However, there was a significant increase (p&amp;lt;0.01) in EAT-resident CD4+ memory T cell populations in pre-existing AF patients and a trend towards significance (p=0.09) in POAF patients. qPCR analysis of immune mediator expression in EAT demonstrated pre-existing AF patients uniquely showed a significant reduction (p&amp;lt;0.005) in the pro-fibrotic mediator transforming growth factor-β (TGF-β) compared to the pre-existing AF patients (graph 1). Conclusions and implications Resident CD4+ memory T cell populations are locally and exclusively elevated in the EAT of pre-existing AF and POAF patients. This would suggest primed antigen-specific CD4+ T cells are present prior to the development of AF in patients who go on to develop de novo POAF. The reduced TGF-β levels observed in the EAT of AF patients indicates the EAT immune mediator profile differs to the underlying myocardial tissue, where increased TGF-β levels have previously been described in the persistent AF cohort. Therapies targeting this EAT-resident CD4+ T cell population would provide a novel approach to the management of the inflammatory components of AF genesis. Graph 1 Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Barts Charity, Abbott

Modulation of experimental autoimmune encephalomyelitis through colonisation of the gut with Escherichia coli.

Multiple sclerosis (MS) is a neuro-inflammatory autoimmune disease of the central nervous system (CNS) that affects young adults. It is characterised by the development of demyelinating lesions and inflammation within the CNS. Although the causes of MS are still elusive, recent work using patient samples and experimental animal models has demonstrated a strong relationship between the gut microbiota and its contribution to CNS inflammation and MS. While there is no cure for MS, alteration of the gut microbiota composition through the use of probiotics is a very promising treatment. However, while most recent works have focused on the use of probiotics to modify pre-existing disease, little is known about its role in protecting from the establishment of MS. In this study, we determined whether colonisation with the probiotic bacterium Escherichia coli strain Nissle 1917 (EcN) could be used as a prophylactic strategy to prevent or alter the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. We found that double gavage (two doses) of EcN before induction of EAE delayed disease onset and decreased disease severity. We also found that EcN-treated mice had decreased amounts of perivascular cuffing, CD4+ T cell infiltration into the CNS, together with significantly decreased absolute numbers of Th1 cells, and reduced activation of microglia. Although further studies are necessary to comprehend the exact protective mechanisms induced, our study supports a promising use of EcN as a probiotic for the prevention of MS.

Quantitative and qualitative alterations of circulating myeloid cells and plasmacytoid DC in SARS-CoV-2 infection.

SARS-CoV-2 is responsible for a new infectious disease (COVID-19) in which individuals can either remain asymptomatic or progress from mild to severe clinical conditions including acute respiratory distress syndrome and multiple organ failure. The immune mechanisms that potentially orchestrate the pathology in SARS-CoV-2 infection are complex and only partially understood. There is still paucity of data on the features of myeloid cells involved in this viral infection. For this reason, we investigated the different activation status profiles and the subset distribution of myeloid cells and their correlation with disease progression in 40 COVID-19 patients at different stages of disease. COVID-19 patients showed a decrease in the absolute number of plasmacytoid and myeloid dendritic cells, different subset distribution of monocytes and different activation patterns of both monocytes and neutrophils, coupled to a significant reduction of HLA-DR monocyte levels. We found that some of these alterations are typical of all COVID-19 patients, while some others vary at different stages of the disease and correlate with biochemical parameters of inflammation. Collectively, these data suggest that not only the lymphoid, but also the myeloid compartment, is severely affected by SARS-CoV-2 infection.

Immunosuppression Has Long-Lasting Effects on Circulating Follicular Regulatory T Cells in Kidney Transplant Recipients.

Background: FoxP3+ follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector immune responses against transplanted tissue.Methods: To understand the biology of Tfr cells in kidney transplant patients treated with tacrolimus and mycophenolate mofetil (MMF) combination immunosuppression, we measured circulating (c)Tfh and cTfr cells in peripheral blood by flow cytometry in n = 211 kidney transplant recipients. At the time of measurement patients were 5–7 years after transplantation. Of this cohort of patients, 23.2% (49/211) had been previously treated for rejection. Median time after anti-rejection therapy was 4.9 years (range 0.4–7 years). Age and gender matched healthy individuals served as controls.Results: While the absolute numbers of cTfh cells were comparable between kidney transplant recipients and healthy controls, the numbers of cTfr cells were 46% lower in immunosuppressed recipients (p < 0.001). More importantly, in transplanted patients, the ratio of cTfr to cTfh was decreased (median; 0.10 vs. 0.06), indicating a disruption of the balance between cTfr and cTfh cells. This shifted balance was observed for both non-rejectors and rejectors. Previous pulse methylprednisolone or combined pulse methylprednisolone + intravenous immunoglobulin anti-rejection therapy led to a non-significant 30.6% (median) and 51.2% (median) drop in cTfr cells, respectively when compared to cTfr cell numbers in transplant patients who did not receive anti-rejection therapy. A history of alemtuzumab therapy did lead to a significant decrease in cTfr cells of 85.8% (median) compared with patients not treated with anti-rejection therapy (p < 0.0001). No association with tacrolimus or MMF pre-dose concentrations was found.Conclusion: This cross-sectional study reveals that anti-rejection therapy with alemtuzumab significantly lowers the number of cTfr cells in kidney transplant recipients. The observed profound effects by these agents might dysregulate cTfr functions.

A single copy of large tumor suppressor 1 or large tumor suppressor 2 is sufficient for normal hematopoiesis.

BackgroundHematopoietic stem cells (HSCs) have the ability to differentiate into all subsets of blood cells and self-renew. Large tumor suppressor 1 (LATS1) and large tumor suppressor 2 (LATS2) kinases are essential for cell cycle regulation, organism fitness, genome integrity, and cancer prevention. Here, we investigated whether Lats1 and Lats2 are critical for the maintenance of the self-renewal and quiescence capacities of HSCs in mice.MethodsQuantitative reverse transcription-polymerase chain reaction was used to determine the expression levels of Lats1 and Lats2 in subsets of progenitor cells and mature bone marrow cells. A clustered regularly interspaced short palindromic repeats system was used to generate Lats1 or Lats2 knockout mice. Complete blood cell counts were used to compare the absolute number of white blood cells, lymphocytes, monocytes, neutrophils, and platelets between Lats1 or Lats2 heterozygotes and littermates. Flow cytometry was used to assess the size of hematopoietic progenitor cells (HPCs) and HSC pools in Lats1 or Lats2 heterozygotes and littermates. The comparison between the two groups was analyzed using Student's t test.ResultsLats1 and Lats2 were widely expressed in hematopoietic cells with higher expression levels in primitive hematopoietic cells than in mature cells. Lats1 or Lats2 knockout mice were generated, with the homozygotes showing embryonic lethality. The size of the HPC and HSC pools in Lats1 (HPC: wild-type [WT] vs. heterozygote, 220,426.77 ± 54,384.796 vs. 221,149.4 ± 42,688.29, P = 0.988; HSC: WT vs. heterozygote, 2498.932 ± 347.856 vs. 3249.763 ± 370.412, P = 0.105) or Lats2 (HPC: WT vs. heterozygote, 425,540.52 ± 99,721.86 vs. 467,127.8 ± 89,574.48, P = 0.527; HSC: WT vs. heterozygote, 4760.545 ± 1518.01 vs. 5327.437 ± 873.297, P = 0.502) heterozygotes were not impaired. Moreover, the depletion of Lats1 or Lats2 did not affect the overall survival of the heterozygotes (Lats1: P = 0.654; Lats2: P = 0.152).ConclusionThese results indicate that a single allele of Lats1 or Lats2 may be sufficient for normal hematopoiesis.